Novel microsphere and method for production thereof

ABSTRACT

A provision of a method for producing a microsphere improved in dispersibility. A method for producing a microsphere improved in dispersibility, characterized in that during the production of microspheres by an in-water drying method, an osmotic pressure regulating agent is added to an outer water phase.

TECHNICAL FIELD

The present invention relates to a microsphere having improveddispersibility, a method of producing the same, a sustained-releasecomposition containing the microsphere, and so on.

BACKGROUND ART

For example, Japanese Patent Application Laid-Open (JP-A) Nos.57-118512, 57-150609 and 6-145046 disclose a method of producing asustained-release microsphere from a W/O type emulsion using abiodegradable polymer.

The sustained-release biodegradable polymer is useful as, for example, abase material for a physiologically active substance-enclosingmicrosphere or the like. Known examples of such a biodegradable polymerinclude polylactic acid and a copolymer of lactic acid and glycolic acid(e.g., JP-A 11-269094).

After produced by any conventional synthesis method, such biodegradablepolymers have been used as they are. It has been found, however, thatsuch unmodified product as synthesized has a low amount of the terminalcarboxyl group and thus can be less useful as a sustained-release basematerial. Thus, investigations have been made on a process including thesteps of hydrolyzing such unmodified biodegradable polymer with a highmolecular weight to form a product having an appropriate weight averagemolecular weight and then using the product as a base material for asustained-release preparation.

However, the product obtained after the hydrolysis and washing withwater can easily cause an initial burst and thus is not suitable as thesustained-release base material, even though it has an appropriateweight average molecular weight and an appropriate amount of theterminal carboxyl group. Thus, there has been a demand for itsimprovement under the present circumstances.

JP-A 7-97334 discloses a sustained-release preparation and a method ofproducing the same, wherein the preparation comprises a physiologicallyactive peptide or a salt thereof and a biodegradable polymer having afree carboxyl group at its terminal.

However, these literatures are silent on any method for improving thedispersibility of the microsphere.

It is therefore an object of the present invention to provide amicrosphere having improved dispersibility, a method of producing thesame, and so on.

DISCLOSURE OF INVENTION

The present inventors have made active investigations with the aboveobject in view and consequently found that in an in-water drying processfor production of microspheres, addition of an osmotic pressureregulating agent to the outer aqueous phase can surprisingly improve thedispersibility of the microsphere product. Based on the finding, thepresent inventors have further made investigations and finally completedthe present invention.

Thus, the present invention provides:

-   (1) a method of producing a microsphere having improved    dispersibility, which comprises adding an osmotic pressure    regulating agent to an outer aqueous phase in producing the    microspheres by an in-water drying method;-   (2) the method according to the above (1), wherein the    dispersibility is improved to such a degree that about 400 to about    700 mg of the microspheres can be dispersed in 1.5 ml of a    dispersion medium for injection in less than two minutes;-   (3) the method according to the above (1), wherein a W/O/W type    emulsion is used in the in-water drying method;-   (4) the method according to the above (3), which further comprises    adding a drug carrier to an inner aqueous phase;-   (5) the method according to the above (1), wherein an O/W type    emulsion is used in the in-water drying method;-   (6) the method according to the above (1), wherein an S/O/W type    emulsion is used in the in-water drying method;-   (7) a method of producing microspheres, which comprises dispersing a    W/O type emulsion in an outer aqueous phase that contains an osmotic    pressure regulating agent, wherein the W/O type emulsion consists of    an inner aqueous phase containing a physiologically active substance    or a salt thereof and an oil phase of a solution containing a lactic    acid polymer with a weight average molecular weight of 15000 to    50000 or a salt thereof; and subjecting the dispersion to an    in-water drying method;-   (8) the method according to the above (7), wherein the content of a    polymer with a weight average molecular weight of 5000 or less in    the lactic acid polymer or the salt thereof is about 10% by weight    or less;-   (9) the method according to the above (7), wherein the content of a    polymer with a weight average molecular weight of 5000 or less in    the lactic acid polymer or the salt thereof is about 5% by weight or    less;-   (10) the method according to the above (7), wherein the content of a    polymer with a weight average molecular weight of 3000 or less in    the lactic acid polymer or the salt thereof is about 1.5% by weight    or less;-   (11) the method according to the above (7), wherein the content of a    polymer with a weight average molecular weight of 1000 or less in    the lactic acid polymer or the salt thereof is about 0.1% by weight    or less;-   (12) the method according to the above (7), wherein the weight    average molecular weight of the lactic acid polymer or the salt    thereof is 15000 to 40000;-   (13) the method according to the above (7), wherein the weight    average molecular weight of the lactic acid polymer or the salt    thereof is 17000 to 26000;-   (14) the method according to the above (1) or (7), wherein the    osmotic pressure regulating agent is alcohol, sugar, amino acid, a    peptide, a protein, a salt of water-soluble amino acid, or a    derivative thereof or a mixture thereof;-   (15) the method according to the above (1) or (7), wherein the    osmotic pressure regulating agent is mannitol;.-   (16) the method according to the above (1) or (7), wherein a    concentration of the osmotic pressure regulating agent in the outer    aqueous phase is a concentration at which the osmotic pressure of    the outer aqueous phase is about 1/50 to about 5 times the osmotic    pressure of isotonic sodium chloride solution;-   (17) the method according to the above (7), wherein the    physiologically active substance is a water-soluble physiologically    active substance;-   (18) the method according to the above (7), wherein the    physiologically active substance is a physiologically active    peptide;-   (19) the method according to the above (7), wherein the    physiologically active substance is an LH—RH derivative;-   (20) the method according to the above (7), wherein the LH—RH    derivative is a peptide represented by the formula:    5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z    wherein Y represents DLeu, DAla, DTrp, DSer(tBu), D2Nal or    DHis(ImBzl) and Z represents NH—C₂H₅ or Gly-NH₂, or a salt thereof;-   (21) a microsphere produced by the method according to the above (1)    or (7);-   (22) a sustained-release composition, comprising the microsphere    according to the above (21);-   (23) the sustained-release composition according to the above (22),    which is for prevention or treatment of prostatic cancer, prostatic    hypertrophy, endometriosis, hysteromyoma, metrofibroma, precocious    puberty, dysmenorrhea, or breast cancer, or for contraception;-   (24) the sustained-release composition according to the above (22),    which is for injection;-   (25) the sustained-release composition according to the above (22),    which further comprises mannitol;-   (26) the sustained-release composition according to the above (22),    which contains about 70% by weight or more of the microsphere in the    total composition;-   (27) a method of preventing or treating prostatic cancer, prostatic    hypertrophy, endometriosis, hysteromyoma, metrofibroma, precocious    puberty, dysmenorrhea, or breast cancer or of contraception, which    comprises administering an effective amount of the sustained-release    composition according to the above (22) to a mammal;-   (28) a method which comprises subjecting an emulsion to in-water    drying in the presence of an osmotic pressure regulating agent in    the outer aqueous phase for producing a microsphere having improved    dispersibility, wherein the emulsion contains a physiologically    active substance or a salt thereof and a polymer; and-   (29) use of an osmotic pressure regulating agent in an outer aqueous    phase in subjecting an emulsion containing a physiologically active    substance or a salt thereof and a polymer to in-water drying for    production of a microsphere having improved dispersibility.

The present invention also provides:

-   (30) a microsphere having improved dispersibility, which comprises a    physiologically active substance or a salt thereof; and a lactic    acid polymer with a weight average molecular weight of 15000 to    50000 in which the content of a polymer with a weight average    molecular weight of 5000 or less is about 5% by weight or less, or a    salt thereof;-   (31) the microsphere according to the above (30), wherein about 400    to about 700 mg of a sustained-release composition containing the    microsphere according to the above (30) can be dispersed in 1.5 ml    of a dispersion medium in less than two minutes;-   (32) the microsphere according to the above (30), which can be    produced by an in-water drying method in the presence of an osmotic    pressure regulating agent in an outer aqueous phase;-   (33) the sustained-release composition according to the above (32),    wherein the in-water drying method is a W/O/W type;-   (34) the sustained-release composition according to the above (32),    wherein the in-water drying method is an O/W type;-   (35) the sustained-release composition according to the above (32),    wherein the in-water drying method is an S/O/W type;-   (36) the microsphere according to the above (30), which is produced    by dispersing a W/O type emulsion in an aqueous phase that contains    an osmotic pressure regulating agent, wherein the W/O type emulsion    comprises an inner aqueous phase containing a physiologically active    substance or a salt thereof and an oil phase of a solution    containing a lactic acid polymer with a weight average molecular    weight of 15000 to 50000 in which the content of a polymer with a    weight average molecular weight of 5000 or less is about 5% by    weight or less, or a salt thereof; and subjecting the dispersion to    an in-water drying method;-   (37) the microsphere according to the above (30), wherein the    content of a polymer with a weight average molecular weight of 3000    or less in the lactic acid polymer is about 1.5% by weight or less;-   (38) the microsphere according to the above (30), wherein the    content of a polymer with a weight average molecular weight of 1000    or less in the lactic acid polymer is about 0.1% by weight or less;-   (39) the microsphere according to the above (30), wherein the lactic    acid polymer has a weight average molecular weight of 15000 to    40000;-   (40) the microsphere according to the above (30), wherein the lactic    acid polymer has a weight average molecular weight of 17000 to    26000;-   (41) the microsphere according to the above (30), wherein the    physiologically active substance is a water-soluble physiologically    active substance;-   (42) the microsphere according to the above (41), wherein the    physiologically active substance is a physiologically active    peptide;-   (43) the microsphere according to the above (41), wherein the    physiologically active substance is an LH—RH derivative;-   (44) the microsphere according to the above (43), wherein the LH—RH    derivative is a peptide represented by the formula:    5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z    wherein Y represents DLeu, DAla, DTrp, DSer(tBu), D2Nal or    DHis(ImBzl) and Z represents NH—C₂H₅ or Gly-NH₂;-   (45) the microsphere according to any one of the above (32) to (36),    wherein a concentration of the osmotic pressure regulating agent in    the outer aqueous phase is a concentration at which the osmotic    pressure of the outer aqueous phase is about 1/50 to about 5 times    the osmotic pressure of isotonic sodium chloride solution;-   (46) the microsphere according to any one of the above (32). to    (36), wherein the osmotic pressure regulating agent is alcohol,    sugar, amino acid, a peptide, a protein, a salt of water-soluble    amino acid, or a derivative thereof or a mixture thereof;-   (47) the microsphere according to the above (43), wherein the    alcohol is polyhydric alcohol or monohydric alcohol;-   (48) the microsphere according to the above (47), wherein the    polyhydric alcohol is glycerin, arabitol, xylitol, adonitol,    mannitol, sorbitol, dulcitol, or a mixture thereof;-   (49) the microsphere according to the above (47), wherein the    monohydric alcohol is methanol, ethanol, isopropyl alcohol, or a    mixture thereof;-   (50) the microsphere according to the above (46), the sugar is a    monosaccharide, a disaccharide, an oligosaccharide, or a derivative    thereof or a mixture thereof;-   (51) the microsphere according to the above (50), wherein the    monosaccharide is arabinose, xylose, ribose, 2-deoxyribose, glucose,    fructose, galactose, mannose, sorbose, rhamnose, or fucose;-   (52) the microsphere according to the above (50), wherein the    disaccharide is maltose, cellobiose, α,α-trehalose, lactose, or    sucrose;-   (53) the microsphere according to the above (50), wherein the    oligosaccharide is maltotriose, raffinose or stachyose;-   (54) the microsphere according to the above (50), wherein the    derivative of the monosaccharide, disaccharide or oligosaccharide is    glucosamine, galactosamine, glucuronic acid, or galacturonic acid;-   (55) the microsphere according to the above (46), wherein the amino    acid is glycine, alanine, valine, leucine, isoleucine,    phenylalanine, tyrosine, tryptophan, serine, threonine, proline,    hydroxyproline, cysteine, methionine, aspartic acid, glutamic acid,    lysine, arginine, or histidine;-   (56) the microsphere according to the above (46), wherein the salt    of the water-soluble amino acid is an acid or alkali metal salt of    glycine, alanine, valine, leucine, isoleucine, phenylalanine,    tyrosine, tryptophan, serine, threonine, proline, hydroxyproline,    cysteine, methionine, aspartic acid, glutamic acid, lysine,    arginine, or histidine;-   (57) the microsphere according to any one of the above (32) to (36),    wherein the osmotic pressure regulating agent is mannitol;-   (58) the microsphere according to the above (30), which further    comprises a drug carrier;-   (59) the microsphere according to the above (58), wherein the drug    carrier is albumin, gelatin, salicylic acid, citric acid, or sodium    ethylenediaminetetraacetate;-   (60) the microsphere according to the above (31), wherein the    dispersion medium is a dispersant, a preservative, an isotonic    agent, or a vegetable oil;-   (61) a method of producing a microsphere having improved    dispersibility, which comprises performing an in-water drying method    in the presence of an osmotic pressure regulating agent in an outer    aqueous phase for improving the dispersibility of the resulting    microspheres;-   (62) a method of producing a microcapsule containing a    physiologically active substance or a salt thereof, and a lactic    acid polymer with a weight average molecular weight of 15000 to    50000 or a salt thereof in which the content of a polymer with a    weight average molecular weight of 5000 or less is about 5% by    weight or less, which comprises performing an in-water drying method    in the presence of an osmotic pressure regulating agent in an outer    aqueous phase;-   (63) the method according to the above (62), wherein the in-water    drying method is a W/O/W type;-   (64) the method according to the above (62), wherein the in-water    drying method is an O/W type;-   (65) the method according to the above (62), wherein the in-water    drying method is an S/O/W type;-   (66) a method of producing a microsphere containing a    physiologically active substance or a salt thereof, and a lactic    acid polymer with a weight average molecular weight of 15000 to    50000 or a salt thereof in which the content of a polymer with a    weight average molecular weight of 5000 or less is about 5% by    weight or less, which comprises dispersing a W/O type emulsion in an    aqueous phase that contains an osmotic pressure regulating agent,    wherein the W/O type emulsion comprises an inner aqueous phase    containing the physiologically active substance or the salt thereof    and an oil phase of a solution containing the lactic acid polymer    with a weight average molecular weight of 15000 to 50000 or a salt    thereof in which the content of a polymer with a weight average    molecular weight of 5000 or less is about 5% by weight or less; and    subjecting the dispersion to an in-water drying method;-   (67) the method according to the above (66), wherein a concentration    of the osmotic pressure regulating agent in the outer aqueous phase    is a concentration at which the osmotic pressure of the outer    aqueous phase is about 1/50 to about 5 times the osmotic pressure of    isotonic sodium chloride solution;-   (68) the method according to the above (66), wherein the inner    aqueous phase further contains a drug carrier;-   (69) a sustained-release composition comprising the microsphere    according to the above (30);-   (70) a sustained-release composition for prevention or treatment of    prostatic cancer, prostatic hypertrophy, endometriosis,    hysteromyoma, metrofibroma, precocious puberty, dysmenorrhea or    breast cancer or for contraception, which comprises the microsphere    according to the above (43);-   (71) the sustained-release composition according to the above (69)    or (70), which is for injection;-   (72) the sustained-release composition according to any one of the    above (69) to (71), which further comprises mannitol;-   (73) the sustained-release composition according to any one of the    above (69) to (72), which contains about 70% by weight or more of    the microsphere in the total composition;-   (74) a method of preventing or treating prostatic cancer, prostatic    hypertrophy, endometriosis, hysteromyoma, metrofibroma, precocious    puberty, dysmenorrhea or breast cancer or of contraception, which    comprises administering an effective amount of the microsphere    according to the above (43) or a sustained-release composition    comprising the microsphere to a mammal;-   (75) a long-term sustained-release microsphere which comprises a    physiologically active substance or a salt thereof and a polymer or    a salt thereof and wherein about 400 to 700 mg of a    sustained-release composition comprising the microsphere can be    dispersed in 1.5 ml of a dispersion medium in less than two minutes;-   (76) the microsphere according to the above (75), wherein the    polymer or a salt thereof is a lactic acid polymer with a weight    average molecular weight of 10000 to 50000 or a salt thereof;-   (77) a sustained-release composition comprising the microsphere    according to the above (75) or (76);-   (78) a method of improving the dispersibility of a microsphere    containing a physiologically active substance or a salt thereof and    a polymer, which comprises performing an in-water drying method in    the presence of an osmotic pressure regulating agent in an outer    aqueous phase in producing the microsphere;-   (79) a method for improving the dispersibility of a microsphere    containing a physiologically active substance or a salt thereof and    a polymer in a composition for injection comprising the microsphere,    which comprises performing an in-water drying method in the presence    of an osmotic pressure regulating agent in an outer aqueous phase    for production of the microcapsule;-   (80) a method of using an osmotic pressure regulating agent in an    outer aqueous phase in an in-water drying method for production of a    microspheres which contains a physiologically active substance or a    salt thereof and a polymer and which has improved dispersibility in    a composition for injection comprising the microsphere;-   (81) use of an osmotic pressure regulating agent in an outer aqueous    phase in an in-water drying method for production of a microsphere    which contains a physiologically active substance or a salt thereof    and a polymer and which has improved dispersibility in a composition    for injection comprising the microsphere; and-   (82) an agent for improving the dispersibility of a microcapsule for    use in an outer aqueous phase for an in-water drying method, which    comprises an osmotic pressure regulating agent.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an electron micrograph showing microspheres of ComparativeExample 1.

FIG. 2 is an electron micrograph showing microspheres of Example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

The physiologically active substance for use in the present inventionhas high hydrophilicity and low n-octanol/water (oil/water) partitionratio. Such low oil/water partition ratio means an n-octanol/watersolubility ratio of preferably 1 or less, more preferably 0.1 or less.

The oil/water partition ratio can be determined by the method describedin Jitsusaburo Samejima, “Butsuri Kagaku Jikkenho (PhysicochemicalExperimental Method)”, published by SHOKABO PUBLISHING Co., Ltd., 1961.Specifically, the method is performed as follows. First, n-octanol and abuffer, pH 5.5 (a 1:1 mixture) are put in a test tube. For example, thebuffer is Sφerensen buffer [Ergeb. Physiol., 12, 393 (1912)], Clark-Lubsbuffer [J. Bact., 2(1), 109, 191 (1917)], Macllvaine buffer [J. Biol.Chem., 49, 183 (1921)], Michaelis buffer (DieWasser-stoffionenkonzentration, p. 186 (1.914)]; Kolthoff buffer[Biochem. Z., 179, 410 (1926)], or the like. An appropriate amount of adrug is put in the test tube. The test tube is then capped and immersedin a thermostatic bath (25° C.) while vigorously shaken frequently. Whenthe drug appears to be dissolved in both liquid layers to reachequilibrium, the liquid mixture is allowed to stand or be centrifuged. Acertain amount of the liquid is then pipetted from each of the upper andlower layers, and analyzed so that the concentration of the drug in eachlayer is determined. The oil/water partition ratio is obtained as theratio of the concentration of the drug in the n-octanol layer to that inthe water layer.

Examples of the physiologically active substance include, but are notlimited to, physiologically active substances, antitumor agents,antibiotics, antipyretic agents, analgesics, anti-inflammatory agents,antitussive expectorants, sedatives, muscle relaxants, antiepilepticagents, antiulcer agents, antidepressants, anti-allergic agents,cardiotonics, antiarrhythmic agents, vasodilators, hypotensivediuretics, antidiabetics, anticoagulants, hemostatics, antitubercularagents, hormone agents, narcotic antagonists, bone resorptionsuppressors, and angiogenesis inhibitors.

Any pharmacologically useful sub-stance may be used as thephysiologically active substance in the present invention, and it may bea non-peptide compound or a peptide compound. The non-peptide compoundmay be an agonist, an antagonist, a compound having an enzyme-inhibitingeffect, or the like. For example, the peptide compound is preferably aphysiologically active peptide, which may have a molecular weight ofabout 300 to 40,000, preferably of about 400 to 30,000, more preferablyof about 500 to 20,000.

Examples of the physiologically active peptide include luteinizinghormone-releasing hormone (LH—RH), insulin, somatostatin, growthhormones, growth hormone-releasing hormone (GH—RH), prolactin,erythropoietin, adrenocortical hormone, melanocyte-stimulating hormone,thyroid hormone-releasing hormone (TRH), thyroid-stimulating hormone,luteinizing hormone, follicle-stimulating hormone, vasopressin,oxytocin, calcitonin, gastrin, secretin, pancreozymin, cholecystokinin,angiotensin, human placental lactogen, human chorionic gonadotropin,enkephalin, endorphin, kyotorphin, tuftsin, thymopoietin, thymosin,thymostimulin, thymic humoral factor, blood thymic factor, tumornecrosis factor, colony-stimulating factors, motilin, dynorphin,bombesin, neurotensin, caerulein, bradykinin, atrial natriuretic factor,nerve growth factor, cell growth factor, neurotrophic factor, peptideshaving endothelin antagonism and derivatives thereof, and fragmentsthereof and derivatives of such fragments.

Preferred examples of the physiologically active peptide include LH—RHderivatives effective against hormone-dependent diseases, especially sexhormone-dependent cancer (such as prostatic cancer, uterus cancer,breast cancer, and pituitary tumor) or sex hormone-dependent diseasessuch as prostatic hypertrophy, endometriosis, hysteromyoma, precociouspuberty, dysmenorrhea, amenorrhea, premenstrual syndrome andmultilocular ovarian syndrome, or effective for contraception (orinfertility, if a rebound effect is used after the drug holiday), orsalts thereof. Additional examples include LH—RH derivatives effectiveagainst benign or malignant tumor that is not sex hormone-dependent butsensitive to LH—RH, or salts thereof.

Specific examples of the LH—RH derivatives or salts thereof includepeptides as disclosed in “Treatment with GnRH analogs: Controversies andperspectives” published by The Parthenon Publishing Group Ltd., 1996,JP-A 3-503165, JP-A 3-101695, JP-A 7-97334 and JP-A 8-259460.

The LH—RH derivative may be an LH—RH agonist or LH—RH antagonist, andexamples of the LH—RH antagonist include physiologically active peptidesrepresented by the general formula [I]:X-D2Nal-D4ClPhe-D3Pal-Ser-A-B-Leu-C-Pro-DAlaNH₂wherein X represents N(4H₂-furoyl)Gly or NAc, A represents a residueselected from NMeTyr, Tyr, Aph(Atz) and NMeAph(Atz), B represents aresidue selected from DLys(Nic), DCit, DLys(AzaglyNic), DLys(AzaglyFur),DhArg(Et₂), DAph(Atz) and DhCi, and C represents Lys(Nisp), Arg orhArg(Et₂), and salts thereof.

Examples of the LH—RH agonist include physiologically active peptidesrepresented by the general formula [II]:5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Zwherein Y represents a residue selected from DLeu, DAla, DTrp,DSer(tBu), D2Nal and DHis(ImBzl), and Z represents NH—C₂H₅ or Gly-NH₂,and salts thereof. Particularly, preferred is a peptide wherein Y isDLeu and Z is NH—C₂H₅ (that is, peptide A represented by the formula:5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH—C₂H₅, leuprorelin) or asalt (such as an acetate) thereof.

These peptides can be produced by or based on the method as disclosed inthe above literatures or publications.

The abbreviations used herein have the following meanings, respectively:Abbreviations: Names N(4H₂-furoyl)Gly: N-tetrahydrofuroylglycine residueNAc: N-acetyl group D2Nal: D-3-(2-naphthyl)alanine residue D4ClPhe:D-3-(4-chloro)phenylalanine residue D3Pal: D-3-(3-pyridyl)alanineresidue NMeTyr: N-methylthyrosine residue Aph(Atz):N-[5′-(3′-amino-1′H-1′,2′,4′- triazolyl)]phenylalanine residueNMeAph(Atz): N-methyl-[5′-(3′-amino-1′H-1′,2′,4′-triazolyl)]phenylalanine residue DLys(Nic): D-(e-N-nicotinoyl)lysineresidue Dcit: D-citrulline residue DLys(AzaglyNic):D-(azaglycylnicotinoyl)lysine residue DLys(AzaglyFur):D-(azaglycylfuranyl)lysine residue DhArg(Et₂):D-(N,N′-diethyl)homoarginine residue DAph(Atz):D-N-[5′-(3′-amino-1′H-1′,2′,4′- triazolyl)]phenylalanine residue DhCi:D-homocitrulline residue Lys(Nisp): (e-N-isopropyl)lysine residuehArg(Et₂): (N,N′-diethyl)homoarginine residue

Abbreviations for other amino acids are according to those defined bythe IUPAC-IUB Commission on Biochemical Nomenclature or defined inEuropean Journal of Biochemistry Vol. 138, pp. 9-37, 1984 or accordingto conventional abbreviations in the field. If not stated otherwise,amino acids are in L-configuration, although they may have opticalisomers.

Examples of the physiologically active peptide also include LH—RHantagonists (see U.S. Pat. Nos. 4,086,219, 4,124,577, 4,253,997, and4,317,815).

Additional examples of the physiologically active peptide includeinsulin, somatostatin, somatostatin derivatives (see U.S. Pat. Nos.4,087,390, 4,093,574, 4,100,117, and 4,253,998), growth hormone,prolactin, adrenocorticotropic hormone (ACTH), melanocyte-stimulatinghormone (MSH), thyroid hormone-releasing hormone (represented by thestructural formula: (Pyr)Glu-His-ProNH2, hereinafter also referred to asTRH) and salts or derivatives thereof (see JP-A 50-121273 and JP-A52-116465), thyroid-stimulating hormone (TSH), luteinizing hormone (LH),follicle-stimulating hormone (FSH), vasopressin, a vasopressinderivative [desmopressin, see Endocrine Journal published by The JapanEndocrine Society, Vol. 54, No. 5, pp. 676-691 (1978)], oxytocin,calcitonin, parathyroid hormone, glucagon, gastrin, secretin,pancreozymin, cholecystokinin, angiotensin, human placental lactogen,human chorionic gonadotropin (HCG), enkephalin, enkephalin derivatives(see U.S. Pat. No. 4,277,394 and European Patent Application Laid-OpenNo. 31567), endorphin, kyotorphin, interferons (such as α-, β- andγ-interferons), interleukins (such as I, II and III), tuftsin,thymopoietin, thymosin, thymostimulin, thymic humoral factor (THF),blood thymic factor (FTS) and derivatives thereof (see U.S. Pat. No.4,229,438), other thymic factors [Igaku no Ayumi, Vol. 125, No. 10, pp.835-843 (1983)], tumor necrosis factor (TNF), colony-stimulating factor(CSF), motilin, dynorphin, bombesin, neurotensin, caerulein, bradykinin,urokinase, asparaginase, kallikrein, substance P, nerve growth factor,cell growth factor, neurotrophic factor, blood coagulation factors VIIIand IX, lysozyme chloride, polymyxin B, colistin, gramicidin,bacitracin, erythropoietin (EPO), and endothelin-antagonistic peptides(see European Patent Application Laid-Open Nos. 436189, 457195 and496452, and JP-A Nos. 3-94692 and 3-130299).

Examples of the antitumor agents include bleomycin, methotrexate,actinomycin D, mitomycin C, binblastin sulfate, bincrystin sulfate,daunorubicin, adriamycin, neocartinostatin, cytosine arabinoside,fluorouracil, tetrahydrofuryl-5-fluorouracil, krestin, picibanil,lentinan, levamisole, bestatin, azimexon, glycyrrhizin, polyI:C,polyA:U, and polyICLC.

Examples of the antibiotics include gentamicin, dibekacin, kanedomycin,lividomycin, tobramycin, amikacin, fradiomycin, sisomycin, tetracyclinehydrochloride, oxytetracycline hydrochloride, rolitetracycline,doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin,cefalothin, cefaloridine, cefotiam, cefsulodin, cefmenoxime,cefmetazole, cefazolin, cefotaxime, cefoperazon, ceftizoxime,moxalactam, thienamycin, sulfazecin, and aztreonam.

Examples of the antipyretic agents, analgesics and anti-inflammatoryagents include salicylic acid, sulpyrine, flufenamic acid, diclofenac,indomethacin, morphine, pethidine hydrochloride, levorphanol tartrate,and oxymorphone.

Examples of the antitussive expectorants include ephedrinehydrochloride, methylephedrine hydrochloride, noscapine hydrochloride,codeine phosphate, dihydrocodeine phosphate, alloclamide hydrochloride,clofedanol hydrochloride, picoperidamine hydrochloride, chloperastine,protokylol hydrochloride, isoproterenol hydrochloride, sulbutamolsulfate, and terbutaline sulfate.

Examples of the sedatives include chlorpromazine, prochlorperazine,trifluoperazine, atropine sulfate, and methylscopolamine bromide.

Examples of the muscle relaxants include pridinol methanesulfonate,tubocurarine chloride and pancuronium bromide.

Examples of the antiepileptic agents include phenytoin, ethosuximide,acetazolamide sodium, and chlordiazepoxide.

Examples of the antiulcer agents include metoclopramide and histidinehydrochloride.

Examples of the antidepressants include imipramine, clomipramine,noxiptiline, and phenelzine sulfate.

Examples of the anti-allergic agents include diphenhydraminehydrochloride, chlorpheniramine maleate, tripelennamine hydrochloride,methdilazine hydrochloride, clemizole hydrochloride, diphenylpyralinehydrochloride, and methoxyphenamine hydrochloride.

Examples of the cardiotonics include trans-n-oxocamphor, theophyllol,aminophylline, and etilefrine hydrochloride.

Examples of the antiarrhythmic agents include propranol, alprenolol,bufetolol, and oxprenolol.

Examples of the vasodilators include oxyfedrine hydrochloride,diltiazem, tolazoline hydrochloride, hexobendine, and bamethan sulfate.

Examples of the hypotensive diuretics include hexamethonium bromide,pentolinium, mecamylamine hydrochloride, ecarazine hydrochloride, andclonidine.

Examples of the antidiabetics include glymidine sodium, glipizide,phenformin hydrochloride, buformin hydrochloride, and metformin.

Examples of the anticoagulants include heparin sodium and sodiumcitrate.

Examples of the hemostatics include thromboplastin, thrombin, menadionesodium hydrogen sulfite, acetomenaphthone, ε-aminocaproic acid,tranexamic acid, carbazochrome sodium sulfonate, and adrenochromemonoaminoguanidine methanesulfonate.

Examples of the antitubercular agents include isoniazid, ethambutol andpara-aminosalicylic acid.

Examples of the hormone agents include predonisolone, predonisolonesodium phosphate, dexamethasone sodium sulfate, betamethasone sodiumphosphate, hexestrol phosphate, hexestrol acetate, and methimazole.

Examples of the narcotic antagonists include levallorphan tartrate,nalorphine hydrochloride and naloxone hydrochloride.

Examples of the bone resorption suppressors include (sulfur-containingalkyl)aminomethylenebisphosphonic acid.

Examples of the angiogenesis inhibitors include angiogenesis-inhibitingsteroids (see Science, Vol. 221, p. 719 (1983)], fumagillin (seeEuropean Patent Application Laid-Open No. 325199), and fumagillolderivatives (see European Patent Application Laid-Open Nos. 357061,359036, 386667 and 415294).

Any physiologically active substance itself or any pharmacologicallyacceptable salt thereof may be used in the present invention.

If the physiologically active substance has a basic group such as anamino group, such a salt may be a salt of an inorganic acid (or a freeinorganic acid) (such as carbonic acid, bicarbonic acid, hydrochloricacid, sulfuric acid, nitric acid, and boric acid) or a salt of anorganic acid (or a free organic acid) (such as succinic acid, aceticacid, propionic acid, and trifluoroacetic acid).

If the physiologically active substance has an acidic group such as acarboxyl group, such a salt may be a salt of an inorganic base (or afree inorganic base) (such as an alkali metal such as sodium andpotassium and an alkaline earth metal such as calcium and magnesium) ora salt of an organic base (or a free organic base) (such as an organicamine such as triethylamine and a basic amino acid such as arginine).The physiologically active peptide may also form a metal complexcompound (such as a copper complex and a zinc complex).

The polymer for use in the present invention is less soluble orinsoluble in water and biocompatible (biodegradable). The wording “lesssoluble in water” means that the polymer has a solubility of not lessthan 0 and not more than about 3% (w/w) in water, preferably asolubility of not less than 0 and not more than about 1% (w/w) in water.

The biodegradable polymer to be used may have a weight average molecularweight of about 10000 to 50000, preferably of about 15000 to 50000, morepreferably of about 15000 to 40000, particularly preferably of about17000 to 26000. The biodegradable polymer may have a dispersibility ofabout 1.2 to 4.0, particularly preferably of about 1.5 to 3.5.

As used herein, the weight average molecular weight and thedispersibility each means a value determined by Gel PermeationChromatography (GPC).

The amount of the polymer to be used depends on the pharmacologicalactivity and the rate and period of release of the physiologicallyactive substance or a salt thereof and the like. For example, thepolymer may be used as a microsphere base material in an amount of about0.5 to 10,000 times (by weight), preferably of about 1 to 100 times (byweight) the amount of the physiologically active substance or a saltthereof.

The polymer is preferably biodegradable, and examples of such a polymerinclude an aliphatic polyester [such as a homopolymer (such as a lacticacid polymer), or a copolymer (such as a lactic acid/glycolic acidcopolymer and a 2-hydroxybutyric acid/glycolic acid copolymer) of two ormore of α-hydroxy acid (such as glycolic acid, lactic acid,2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyricacid, 2-hydroxycaproic acid, 2-hydroxyisocaproic acid and2-hydroxycaprylic acid), a cyclic dimer of α-hydroxy acid (such asglycolide and lactide), hydroxydicarboxylic acid (such as malic acid) ora hydroxytricarboxylic acid (such as citric acid), or any mixture of thehomopolymer(s) and/or the copolymer(s) (such as a mixture of the lacticacid polymer and the 2-hydroxybutyric acid/glycolic acid copolymer)];poly-α-cyanoacrylate ester, polyamino acid (such aspoly-γ-benzyl-L-glutamic acid, poly-L-alanine, andpoly-γ-methyl-L-glutamic acid), and a maleic anhydride copolymer (suchas a styrene/ maleic acid copolymer). Preferred are the aliphaticpolyester and the poly-α-cyanoacrylate ester. The aliphatic polyester isparticularly preferred.

Preferred examples of the aliphatic polyester include the homopolymer ofα-hydroxy acids or the cyclic dimers of α-hydroxy acid, the copolymer oftwo or more thereof, and the mixture of the homopolymer(s) and/or thecopolymer(s). Particularly preferred is the homopolymer or copolymer ofα-hydroxy acids or the mixture of the homopolymer(s) and/or thecopolymer(s).

If the α-hydroxy acids, the cyclic dimers of the α-hydroxy acid, thehydroxydicarboxylic acids, or the hydroxytricarboxylic acids has anyoptically active center in its molecule, it may be in any of D-, L- andDL-configurations.

The aliphatic polyester may be produced by any known method (forexample, see JP-A 61-28521) without any difficulty. The polymerizationmay be any of random, block and graft types.

The aliphatic polyester may have a weight average molecular weight ofabout 10000 to 50000, preferably of about 15000 to 50000, morepreferably of about 15000 to 40000, particularly preferably of about17000 to 26000. The aliphatic polyester preferably has a dispersibilityof about 1.2 to 4.0, particularly preferably of about 1.5 to 3.5.

If the aliphatic polyester is a lactic acid/glycolic acid copolymer, thecomposition ratio is preferably from about 100/0 to about 50/50 (byweight). If the aliphatic polyester is a 2-hydroxybutyric acid/glycolicacid copolymer, the composition ratio is preferably from about 100/0 toabout 25/75 (by weight).

The lactic acid polymer, the lactic acid/glycolic acid copolymer, or the2-hydroxybutyric acid/glycolic acid copolymer preferably has a weightaverage molecular weight of about 15000 to 50000, particularlypreferably of about 15000 to 40000.

If the aliphatic polyester is a mixture of a lactic acid polymer (A) anda glycolic acid/2-hydroxybutyric acid copolymer (B), for example, themixture ratio represented by (A)/(B) may be from about 10/90 to about90/10 (by weight), preferably from about 25/75 to about 75/25 (byweight).

The lactic acid polymer preferably has a weight average molecular weightof about 15000 to 50000, particularly preferably of about 15000 to40000.

The glycolic acid/2-hydroxybutyric acid copolymer is preferably composedof 40 to 70 moles of glycolic acidm and the remainder (60 to 30 moles)of 2-hydroxybutyric acid. The glycolic acid/2-hydroxybutyric acidcopolymer preferably has a weight average molecular weight of about15000 to 50000, particularly preferably of about 15000 to 40000.

In particular, the polymer to be used in the present invention ispreferably a lactic acid polymer (hereinafter, such a lactic acidpolymer is also simply referred to as the lactic acid polymer of thepresent invention). Examples of the lactic acid polymer include apolymer consisting of only lactic acid and a copolymer of lactic acidand any other monomer (such as glycolic acid). In such a polymer, thecontent of a polymer with a weight average molecular weight of 5000 orless is generally about 10% by weight or less, preferably, the contentof a polymer with a weight average molecular weight of 5000 or less isgenerally about 5% by weight or less; more preferably, the content of apolymer with a weight average molecular weight of 3000 or less is about1.5% by weight or less; still preferably, the content of a polymer witha weight average molecular weight of 1000 or less is about 0.1% byweight or less; still more preferably, the content of a polymer with aweight average molecular weight of 5000 or less is about 5% by weight orless, and the content of a polymer with a weight average molecularweight of 3000 or less is about 1.5% by weight or less; most preferably,the content of a polymer with a weight average molecular weight of 5000or less is about 5% by weight or less, the content of a polymer with aweight average molecular weight of 3000 or less is about 1.5% by weightor less, and the content of a polymer with a weight average molecularweight of 1000 or less is about 0.1% by weight or less.

The lactic acid polymer of the present invention generally has a weightaverage molecular weight of 15000 to 50000, preferably of 15000 to40000, more preferably of 17000 to 26000, particularly preferably of17500 to 25500.

A high molecular weight lactic acid polymer for use as material for thelactic acid polymer of the present invention may be commerciallyavailable or may be produced by any known polymerization method andgenerally has a weight average molecular weight of 15000 to 500000,preferably of 20000 to 100000. Examples of the known polymerizationmethod include condensation polymerization of lactic acid and optionallyglycolic acid, ring-opening polymerization of, for example, lactide andoptionally glycolide with a Lewis acid such as diethyl zinc,triethylaluminum and tin octylate or a catalyst such as a metal salt,ring-opening polymerization of lactide in the presence of ahydroxycarboxylic acid derivative with the protected carboxyl group, inaddition to the conditions of the above mentioned ring-openingpolymerization (for example, International Patent PublicationWO00/35990), ring-opening polymerization of lactide with a catalystunder heating (for example, J. Med. Chem., 16, 897 (1973)), andcopolymerization of lactide and glycolide.

The type of the polymerization may be bulk polymerization in whichlactide or the like is melted and subjected to polymerization reactionor solution polymerization in which lactide or the like is dissolved ina suitable solvent and subjected to polymerization reaction. A polymerproduced by the solution polymerization is industrially preferred foruse as material for the lactic acid polymer of the present invention.

Examples of a solvent for dissolving lactide during the solutionpolymerization include aromatic hydrocarbons such as benzene, tolueneand xylene, decalin, and dimethylformamide.

The resulting lactic acid polymer with a high molecular weight may behydrolyzed by any known hydrolysis method. For example, the lactic acidpolymer with the high molecular weight is dissolved in a suitablesolvent and then water and optionally an acid are added thereto to leadto hydrolysis.

The solvent for dissolving the high molecular weight lactic acid polymermay be any solvent, as long as the lactic acid polymer can be dissolvedin such a solvent in an amount of not more than 10 times (by weight) theamount of the polymer. Specific examples of such a solvent include ahalogenated hydrocarbon such as chloroform and dichloromethane; anaromatic hydrocarbon such as toluene, o-xylene, m-xylene and p-xylene;cyclic ether such as tetrahydrofuran; acetone; andN,N-dimethylformamide. If the solvent used in the preparation of thehigh molecular weight lactic acid polymer is also applicable to thehydrolysis of the polymer, the polymerization and the hydrolysis may becontinuously performed without isolation of the high molecular weightlactic acid polymer.

The amount of the solvent to be used for dissolving the high molecularweight lactic acid polymer is generally 0.1 to 100 times, preferably 1to 10 times the amount of the lactic acid polymer to be a solute.

The amount of water to be added is generally 0.001 to 1 time (byweight), preferably of 0.01 to 0.1 times (by weight) the amount of thehigh molecular weight lactic acid polymer.

Examples of the acid to be optionally added include inorganic acid suchas hydrochloric acid, sulfuric acid and nitric acid; and organic acidsuch as lactic acid, acetic acid and trifluoroacetic acid, and lacticacid is preferred.

The amount of such an acid to be added is generally 0 to 10 times (byweight), preferably of 0.1 to 1 time (by weight) the amount of the highmolecular weight lactic acid polymer.

The reaction temperature of the hydrolysis is generally from 0 to 150°C., preferably from 20 to 80° C.

The reaction time of the hydrolysis may vary depending on the weightaverage molecular weight of the high molecular weight lactic acidpolymer and the reaction temperature, and is generally from 10 minutesto 100 hours, preferably from 1 to 20 hours.

The timing of stopping the hydrolysis process may be determined based onthe weight average molecular weight of the hydrolysis product.Specifically, samples are taken at any appropriate time during thehydrolysis process, the weight average molecular weight of thehydrolysis product in the sample is measured by Gel PermeationChromatography (GPC), and then the hydrolysis process is stopped if themolecular weight is determined as being from about 15000 to 50000,preferably from about 15000 to 30000, more preferably from about 17000to 26000, particularly preferably from 17500 to 25500.

After the above described hydrolysis of the high molecular weight lacticacid polymer, the desired lactic acid polymer may be precipitated fromthe resulting solution which contains the hydrolysis product. Forexample, the hydrolysis product-containing solution is brought intocontact with a solvent that can induce precipitation of the desiredlactic acid polymer.

In a preferred aspect, the hydrolysis product-containing solution is a10 to 50 wt % solution of the lactic acid polymer with a weight averagemolecular weight of 15000 to 50000, preferably of 15000 to 30000, morepreferably of 17000 to 26000, particularly preferably of 17500 to 25500in a solvent capable of dissolving a high molecular weight lactic acidpolymer, for example, halogenated hydrocarbon such as chloroform anddichloromethane; aromatic hydrocarbon such as toluene, o-xylene,m-xylene and p-xylene; cyclic ether such as tetrahydrofuran; acetone;N,N-dimethylformamide; dichloromethane; or xylene.

Examples of a solvent with which the desired lactic acid polymer can beprecipitated from the hydrolysis product-containing solution includealcohols such as methanol and ethanol, chain ethers such as isopropylether, aliphatic hydrocarbons such as hexane, and water.

The amount of the solvent to be used in order to precipitate the desiredlactic acid polymer is generally 0.1 to 100 times (by weight),preferably of 1 to 10 times (by weight) the amount of a solvent of thehydrolysis product-containing solution.

Preferred combination of the type and amount of each solvent may be acombination of a hydrolysis product-containing solution in whichdichloromethane in an amount of 1 to 5 times (by weight) the amount ofthe solute is used and isopropyl ether as a solvent for reducing thesolubility in an amount of 2 to 10 times (by weight) the amount of thedichloromethane.

When the hydrolysis product-containing solution is contacted with thesolvent for precipitating the desired lactic acid polymer, thetemperature of the solvent is generally set at −20 to 60° C., preferably0 to 40° C., and the temperature of the hydrolysis product-containingsolution is generally set at 0 to 40° C., preferably 10 to 30° C.

Examples of a method for contacting the solvent with the hydrolysisproduct-containing solution include a method of adding the hydrolysisproduct-containing solution to the solvent at a time, a method of addingthe hydrolysis product-containing solution dropwise to the solvent, amethod of adding the solvent to the hydrolysis product-containingsolution at a time, and a method of adding the solvent dropwise to thehydrolysis product-containing solution.

The lactic acid polymer of the present invention obtained as shown aboveis preferably used as a base material for a sustained-releasepreparation, because the amount of its terminal carboxyl group is in apreferred range for such a base material.

And, examples of the biocompatible polymer include polystyrene,polymethacrylic acid, a copolymer of acrylic acid and methacrylic acid,polyamino acid, dextran stearate, ethyl cellulose, acetyl cellulose,nitrocellulose, a maleic anhydride-based copolymer, an ethylenevinylacetate-based One of these polymers may be used alone, or two ormore thereof may be used in the form of a copolymer or a simple mixture,or any salt thereof may be used.

The concentration of the polymer in an oil phase may be from about 0.5to about 90% (w/w), preferably from about 2 to about 60% (w/w)

Examples of the drug carrier to be used in the present invention includealbumin, gelatin, citric acid, salicylic acid, sodiumethylenediaminetetraacetate, dextrin, sodium hydrogen sulfite, polyolcompounds such as polyethylene glycol, agar, alginic acid, polyvinylalcohol, and a basic amino acid.

The microsphere of the present invention may be produced by an in-waterdrying method, preferably by a (W/O)/W type, O/W type or S/O/W typein-water drying method.

The (W/O)/W type in-water drying method may comprise preparing a W/Otype emulsion that consists of an inner aqueous phase of a liquidcontaining a physiologically active substance or a salt thereof and anoil phase of a solution containing a polymer; dispersing the emulsioninto an aqueous phase containing an osmotic pressure regulating agent toprepare a (W/O)/W type emulsion; and subjecting the emulsion to anin-water drying method to remove a solvent from the oil phase so thatmicrospheres are produced which contain the physiologically activesubstance or a salt thereof and the polymer.

The O/W type in-water drying method may comprise dispersing an oil phaseinto an aqueous phase containing an osmotic pressure regulating agent toprepare an O/W type emulsion, wherein the oil phase comprises aphysiologically active substance or a salt thereof and a polymer; andsubjecting the emulsion to an in-water drying method to remove a solventfrom the oil phase so that microspheres are produced which contain thephysiologically active substance or a salt thereof and the polymer.

The S/O/W type in-water drying method may comprise dispersing aphysiologically active substance or a salt thereof into an oil phase ofa solution containing a polymer; dispersing the dispersion into anaqueous phase containing an osmotic pressure regulating agent to preparean S/O/W type emulsion; and subjecting the emulsion to an in-waterdrying method to remove a solvent from the oil phase so thatmicrospheres are produced which contain the physiologically activesubstance or a salt thereof and the polymer.

The osmotic pressure regulating agent to be used in the presentinvention may be any substance capable of producing an osmotic pressurein an aqueous solution.

Examples of the osmotic pressure regulating agent include alcohols suchas polyhydric alcohol and monohydric alcohol; sugars such asmonosaccharide, disaccharide and oligosaccharide; water-soluble aminoacids, peptides or proteins; salts of water-soluble amino acid; andderivatives thereof.

Examples of the polyhydric alcohol include trihydric alcohols such asglycerin; pentahydric alcohols such as arabitol, xylitol and adonitol;and hexahydric alcohols such as mannitol, sorbitol and dulcitol. Thehexahydric alcohols are preferred, and mannitol is particularlypreferred.

Examples of the monohydric alcohol include methanol, ethanol andisopropyl alcohol, and ethanol is preferred.

Examples of the monosaccharides include pentoses such as arabinose,xylose, ribose and 2-deoxyribose; and hexoses such as glucose, fructose,galactose, mannose, sorbose, rhamnose and fucose. The hexoses areparticularly preferred.

Examples of the disaccharides include maltose, cellobiose,α,α-trehalose, lactose and sucrose. In particular, lactose and sucroseare preferred.

Examples of the oligosaccharides include trisaccharides such asmaltotriose and raffinose; and tetrasaccharides such as stachyose. Thetrisaccharides are particularly preferred.

Examples of the derivatives of monosaccharides, disaccharides oroligosaccharides include glucosamine, galactosamine, glucuronic acid andgalacturonic acid.

The above amino acid may be any L-form amino acid. Examples of such anamino acid include neutral amino acid such as glycine, alanine, valine,leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine,threonine, proline, hydroxyproline, cysteine and methionine; acidicamino acid such as aspartic acid and glutamic acid; and basic amino acidsuch as lysine, arginine and histidine. Glycine, leucine or arginine ispreferably used, and L-arginine is particularly preferred. Alsoapplicable is a salt of the water-soluble amino acid such as an acidsalt of the water-soluble amino acid (such as a salt of hydrochloricacid, sulfuric acid, phosphoric acid or the like) and an alkali salt ofthe water-soluble amino acid (such as a salt of an alkali metal such assodium and potassium or the like).

Examples of the water-soluble peptides, proteins or derivatives thereofinclude casein, globulin, prolamin, albumin, and gelatin.

One or more of these osmotic pressure regulating agents may be usedalone or in combination.

The amount of the physiologically active substance or a salt thereof tobe used may vary depending on the type of the drug, the desiredpharmacological effect, the desired effect duration, or the like. Forexample, the concentration of the physiologically active substance or asalt thereof in an inner aqueous phase is from about 0.001% to about 90%(w/w), more preferably from about 0.01% to about 80% (w/w), particularlypreferably from about 0.01% to about 70% (w/w).

The osmotic pressure regulating agent may be used at such aconcentration that an outer aqueous phase has an osmotic pressure ofabout 1/50 to about 5 times, preferably of about 1/25 to about 3 times,more preferably of about 1/12 to about 2 times the osmotic pressure ofisotonic sodium chloride solution.

Specifically, if the osmotic pressure regulating agent is a nonionicsubstance, the concentration in an outer aqueous phase is from about0.01% to about 60% (w/w), preferably from about 0.01% to about 40%(w/w), more preferably from about 0.05% to about 30% (w/w), particularlypreferably from about 0.5% to about 1.5% (w/w). If the osmotic pressureregulating agent is an ionic substance, the concentration may becalculated by dividing the above concentration by its total ionicvalence. The concentration of the added osmotic pressure regulatingagent does not have to be equal to or lower than its solubility, and itmay be partially in a dispersed state.

According to the present invention, the addition of the osmotic pressureregulating agent to an outer aqueous phase can provide improveddispersibility of the microsphere product. The degree of suchimprovement is not particularly limited, but preferred is, for example,such a degree that about 400 to 700 mg of the microspheres can bedispersed in 1.5 ml of a dispersion medium for injection in less than 2minutes.

Hereinafter, a method of producing microspheres by a (W/O)/Wtype-in-water drying method according to the present invention isexplained.

In the process as described below, the following ingredients may beadded to an inner aqueous phase as needed:

-   -   (1) Drug carrier: albumin, gelatin, citric acid, salicylic acid,        sodium ethylenediaminetetraacetate, dextrin, sodium hydrogen        sulfite, polyol compounds such as polyethylene glycol, agar,        alginic acid, polyvinyl alcohol, basic amino acid, or the like;    -   (2) pH regulator for keeping the stability and solubility of a        physiologically active substance or a salt thereof: carbonic        acid, acetic acid, oxalic acid, citric acid, phosphoric acid,        hydrochloric acid, sodium hydroxide, arginine, lysine, a salt        thereof, or the like;    -   (3) Stabilizer for a physiologically active substance or the        salt thereof: albumin, gelatin, citric acid, sodium        ethylenediaminetetraacetate, dextrin, sodium hydrogen sulfite,        polyol compounds such as polyethylene glycol, or the like;    -   (4) Preservative: para-hydroxybenzoate esters (such as methyl        paraben and propyl paraben), benzyl alcohol, chlorobutanol,        thimerosal, or the like.        (I) O/W Method

For this method, first, a solution of the polymer in an organic solventis prepared. The organic solvent for use in production of themicrospheres of the present invention preferably has a boiling point of120° C. or lower.

Examples of such an organic solvent include halogenated hydrocarbon(such as dichloromethane, chloroform, dichloroethane, trichloroethane,and carbon tetrachloride), ether (such as ethyl ether and isopropylether), fatty acid ester (such as ethyl acetate and butyl acetate),aromatic hydrocarbon (such as benzene, toluene and xylene), alcohol(such as ethanol and methanol), and acetonitrile. Preferred ishalogenated hydrocarbon, and more preferred is dichloromethane. Theorganic solvent may be any mixture of the above-mentioned solvents inthe appropriate ratio. In such a case, a mixture of halogenatedhydrocarbon and alcohol is preferred, and a mixture of dichloromethaneand ethanol is more preferred.

The concentration of the polymer in the organic solvent solution mayvary depending on the molecular weight of the polymer or the type of theorganic solvent. In a case where dichloromethane is used as the organicsolvent, for example, such a concentration is selected from the range ofgenerally from about 0.5 to about 70% by weight, more preferably fromabout 1 to about 60% by weight, particularly preferably from about 2 toabout 50% by weight.

In a case where a mixture of dichloromethane and ethanol is used as theorganic solvent, the content of ethanol in the mixture solvent isselected from the range of generally from about 0.01 to about 50% (v/v),more preferably from about 0.05 to about 40% (v/v), particularlypreferably from about 0.1 to about 30% (v/v).

To the solution of the polymer in an organic solvent thus obtained, thephysiologically active substance or a salt thereof is added and thendissolved or dispersed. In this process, the physiologically activesubstance or a salt thereof is added in such an amount that the weightratio of the physiologically active substance or a salt thereof to thepolymer is not more than about 1:1, preferably about 1:2.

The resulting solution of a composition comprising the physiologicallyactive substance or a salt thereof and the polymer in an organic solventis then added to an aqueous phase to form an O(oil phase)/W(aqueousphase)-type emulsion. Thereafter, the solvent is evaporated from the oilphase so that microspheres are prepared. In this process, the volume ofthe aqueous phase is selected from the range of generally from about 1time to about 10,000 times, more preferably from about 5 times to about50,000 times, particularly preferably from about 10 times to about 2,000times the volume of the oil phase.

Besides the osmotic pressure regulating agent, an emulsifier may also beadded to the outer aqueous phase. Such an emulsifier may be anyemulsifier capable of forming a stable O/W-type emulsion. Specificexamples of such an emulsifier include an anionic surfactant (such assodium oleate, sodium stearate and sodium lauryl sulfate), a nonionicsurfactant [such as polyoxyethylene sorbitan fatty acid ester (such asTween 80 and Tween 60, Atlas Powder Company) and polyoxyethylene castoroil derivative (such as HCO-60 and HCO-50, Nikko Chemicals)),polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose,lecithin, gelatin, and hyaluronic acid. One or more of theabove-mentioned emulsifiers may be used alone or in combination. It ispreferably used in a concentration of about 0.01 to 10% by weight, morepreferably of about 0.05 to about 5% by weight.

The organic solvent may be removed by a known method or a modifiedmethod based on the known method. Examples of such a method include amethod comprising evaporating the organic solvent under normalatmospheric pressure or gradually reduced pressure while stirring with apropeller stirrer, a magnetic stirrer or the like, and a methodcomprising evaporating the organic solvent under a regulated vacuum witha rotary evaporator or the like.

The microspheres thus obtained are collected by centrifugation orfiltration, washed with distilled water several times to remove the freephysiologically active substance, the emulsifier and the like adhered tothe surfaces of the microspheres, dispersed in distilled water or thelike again, and then lyophilized.

During the process of producing the microspeheres, an antiflocculant maybe added for preventing flocculation of the particles. Examples of suchan antiflocculant include mannitol, lactose, glucose, water-solublepolysaccharide such as starch (such as cornstarch), amino acid such asglycine, and protein such as fibrin and collagen. In particular,mannitol is preferred.

The antiflocculant such as mannitol is generally added in an amount of 0to about 24% by weiaht based on the total amount of the microspheres.

After the lyophilization, if desired, water and the organic solvent maybe removed from the microspheres under reduced pressure by heating undersuch conditions that the microspheres are not fused with each other.Preferably, the microspheres are heated at about the intermediate glasstransition temperature of the polymer, which is determined with adifferential scanning calorimeter under the condition of the temperaturerising rate of 10° C. to 20° C. per minute, or a slightly highertemperature than the intermediate glass transition temperature. Morepreferably, the microspheres are heated at about the intermediate glasstransition temperature of the polymer to about 30° C. higher temperaturethan the glass transition temperature. Particularly when the polymer isa lactic acid-glycolic acid polymer, the microspheres are preferablyheated at about its intermediate glass transition temperature to about10° C. higher temperature than the intermediate glass transitiontemperature, more preferably at about its intermediate glass transitiontemperature to about 5° C. higher temperature than the intermediateglass transition temperature.

The heating time may vary depending on the amount of the microspheres orthe like. It is generally from about 12 hours to about 168 hours,preferably from about 24 hours to about 120 hours, particularlypreferably from about 48 hours to about 96 hours, after the microspheresreach the desired temperature.

A method for heating the microspheres may be any method capable ofheating a population of microspheres uniformly and is not particularlylimited.

Examples of the heat drying method include a method of heat drying in aconstant-temperature bath, a fluidized-bed bath, a mobile bath or a kilnand a method of heat drying with a microwave. Preferred is a method ofheat drying in a constant-temperature bath.

(II) W/O/W Method

First, a solution of the polymer in an organic solvent is prepared.

Examples of the organic solvent include halogenated hydrocarbon (such asdichloromethane, chloroform, dichloroethane, trichloroethane, and carbontetrachloride), ether (such as ethyl ether and isopropyl ether), fattyacid ester (such as ethyl acetate and butyl acetate), aromatichydrocarbon (such as benzene, toluene and xylene), alcohol (such asethanol and methanol), and acetonitrile. Preferred is halogenatedhydrocarbon, and more preferred is dichloromethane. The organic solventmay be any mixture of the above-mentioned solvents in the appropriateratio. In such a case, a mixture of halogenated hydrocarbon and alcoholis preferred, and a mixture of dichloromethane and ethanol is morepreferred.

The concentration of the polymer in the organic solvent solution mayvary depending on the molecular weight of the polymer or the type of theorganic solvent. In a case where dichloromethane is used as the organicsolvent, for example, such a concentration is selected from the range ofgenerally from about 0.5 to about 70% by weight, more preferably fromabout 1 to about 60% by weight, particularly preferably from about 2 toabout 50% by weight.

To the solution of the polymer in an organic solvent (an oil phase), thephysiologically active substance, a salt thereof or a solution of thesalt [wherein the solvent is water or a mixture of water and alcohol(such as methanol or ethanol)] is then added. The resulting mixture isemulsified by any known method such as with a homogenizer or sonicationto form a W/O-type emulsion.

The resulting W/O-type emulsion comprising the physiologically activesubstance or a salt thereof and the polymer is then added to an aqueousphase to form a W(inner aqueous phase)/O(oil phase)/W(outer aqueousphase)-type emulsion. Thereafter, the solvent is evaporated from the oilphase so that microspheres are prepared. In this process, the volume ofthe outer aqueous phase is selected from the range of generally fromabout 1 time to about 10,000 times, more preferably from about 5 timesto about 50,000 times, particularly preferably from about 10 times toabout 2,000 times the volume of the oil phase.

The osmotic pressure regulating agent and emulsifier that may beoptionally added to the outer aqueous phase and the subsequentpreparation method may be the same as described in the above section(I).

An emulsifier may be added to the outer aqueous phase. Such anemulsifier may be any emulsifier capable of forming a stable O/W-typeemulsion. Specific examples of such an emulsifier include an anionicsurfactant (such as sodium oleate, sodium stearate and sodium laurylsulfate), a nonionic surfactant [such as polyoxyethylene sorbitan fattyacid ester (such as Tween 80 and Tween 60, Atlas Powder Company) andpolyoxyethylene castor oil derivative (such as HCO-60 and HCO-50, NikkoChemicals)], polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, and hyaluronic acid. One or more of theabove-mentioned emulsifiers may be used alone or in combination. It ispreferably used in a concentration of about 0.01 to 10% by weight, morepreferably of about 0.05 to about 5% by weight.

The organic solvent may be removed by a known method or a modifiedmethod based on the known method. Examples of such a method include amethod comprising evaporating the organic solvent under normalatmospheric pressure or gradually reduced pressure while stirring with apropeller stirrer, a magnetic stirrer, an ultrasonic generator or thelike, a method comprising evaporating the organic solvent under aregulated vacuum with a rotary evaporator or the like, and a methodcomprising gradually removing the organic solvent using a dialysismembrane.

The microspheres thus obtained are collected by centrifugation orfiltration, washed with distilled water several times to remove the freephysiologically active substance or a salt thereof, the drug carrier,the emulsifier and the like adhered to the surfaces of the microspheres,dispersed in distilled water or the like again, and then lyophilized.

During the process of producing the microspeheres, an antiflocculant maybe added for preventing flocculation of the particles. Examples of suchan antiflocculant include mannitol, lactose, glucose, water-solublepolysaccharide such as starch (such as cornstarch), amino acid such asglycine, and protein such as fibrin and collagen. In particular,mannitol is preferred.

The antiflocculant such as mannitol is generally added in an amount of 0to about 24 by weight based on the total amount of the microspheres.

After the lyophilization, if desired, water and the organic solvent maybe removed from the microspheres under reduced pressure by heating undersuch conditions that the microspheres are not fused with each other.Preferably, the microspheres are heated at about the intermediate glasstransition temperature of the polymer, which is determined with adifferential scanning calorimeter under the condition of the temperaturerising rate of 10° C. to 20° C. per minute, or a slightly highertemperature than the intermediate glass transition temperature. Morepreferably, the microspheres are heated at about the intermediate glasstransition temperature of the polymer to about 30° C. higher temperaturethan the glass transition temperature. Particularly when the polymer isa lactic acid-glycolic acid polymer, the microspheres are preferablyheated at about its intermediate glass transition temperature to about10° C. higher temperature than the intermediate glass transitiontemperature, more preferably at about its intermediate glass transitiontemperature to about 5° C. higher temperature than the intermediateglass transition temperature.

The heating time may vary depending on the amount of the microspheres orthe like. It is generally from about 12 hours to about 168 hours,preferably from about 24 hours to about 120 hours, particularlypreferably from about 48 hours to about 96 hours, after the microspheresreach the desired temperature.

A method for heating the microspheres may be any method capable ofheating a population of microspheres uniformly and is not particularlylimited.

Examples of the heat drying method include a method of heat drying in aconstant-temperature bath, a fluidized-bed bath, a mobile bath or a kilnand a method of heat drying with a microwave. Preferred is a method ofheat drying in a constant-temperature bath.

The microsphere of the present invention produced by the method of thepresent invention refers to an injectable spherical fine particle whichcan be dispersed in a solution. For example, its shape and form can bedetermined by observation with a scanning electron microscope. Themicrosphere may be in the form of a microcapsule or a microparticle, andthe microcapsule is preferred.

The weight content of the physiologically active substance or a saltthereof in the microsphere of the present invention may vary dependingon the type of the physiologically active substance or a salt thereof,the desired pharmacological effect, the desired effect duration, and thelike. For example, when the physiologically active substance or a saltthereof is a physiologically active peptide or a salt thereof, thecontent may be from about 0.001 to about 50% by weight, preferably fromabout 0.02 to about 40% by weight, more preferably from about 0.1 toabout 30% by weight, still more preferably about 0.1 to about 24% byweight, most preferably from about 3 to about 24% by weight, based onthe total weight of the microsphere. When the physiologically activesubstance or a salt thereof is a non-peptidic physiologically activesubstance or a salt thereof, the content may be from about 0.01 to about80% by weight, preferably from about 0.1 to about 50% by weight.

The weight content of the polymer in the microsphere of the presentinvention may be from about 50 to about 100% by weight, preferably fromabout 70 to about 100% by weight, more preferably from about 85 to about95% by weight, based on the total weight of the microsphere.

The weight content of the drug carrier in the microsphere of the presentinvention may be from about 0.01 to about 50% by weight, preferably fromabout 0.1 to about 30% by weight, more preferably from about 5 to about15% by weight, based on the total weight of the microsphere.

The microsphere of the present invention has few small pores on thesurface and has a good dispersibility in a suspension for injection.

Since the microsphere of the present invention has such a gooddispersibility, a large amount of the microspheres can be suspended in asuspension for injection. Thus, a suspension for injection caneventually contain a large amount of the physiologically activesubstance or a salt thereof, even if the microsphere does not contain adrug carrier such as hydroxynaphthoic acid.

The microsphere of the present invention may be administered, as it isor after formulation into various dosage forms, as an injection orimplant for muscle, subcutis, organ or the like, a transmucosal agentfor nasal cavity, rectum, uterus or the like, or an oral agent (such asa capsule (such as a hard capsule and a soft capsule), a solidpreparation such as a granule and a powder, and a liquid preparationsuch as a syrup, an emulsion and a suspension) or the like.

For example, the microspheres of the present invention may be mixed witha dispersion medium such as a dispersing agent (such as a surfactantsuch as Tween 80 and HCO-60; and polysaccharide such as sodiumhyaluronic acid, carboxymethylcellulose and sodium alginate), apreservative (such as methyl paraben and propyl paraben), and anisotonic agent (such as sodium chloride, mannitol, sorbitol, glucose,and proline) to prepare an aqueous suspension, or mixed with adispersion medium such as a vegetable oil such as sesame oil and cornoil to prepare an oily suspension, so that a practical sustained-releaseinjection can be prepared.

The particle diameters of the microspheres of the present invention foruse in the suspension injection should be in such a range that they havea satisfactory dispersibility and a satisfactory ability to pass througha needle. For example, the microspheres have an average particlediameter of about 0.1 to 300 μm, preferably of about 0.5 to 150 μm, morepreferably from about 1 to 100 μm.

The microspheres of the present invention may be formulated into asterile preparation by any method including, but not limited to, sterileconditions during all preparation steps, sterilization with gammaradiation and addition of an antiseptic.

For the above sustained-release microsphere injection, an excipient(such as mannitol, sorbitol, lactose, and glucose) may be added to theabove components of the suspension, and the suspension may bere-dispersed and then freeze-dried or spray-dried to obtain a solid. Atthe time of administration, distilled water for injection or anyappropriate dispersion medium may be added to the solid to prepare amore stable sustained-release injection.

In a case where an excipient such as mannitol is added to thesustained-release microsphere injection, the content of the excipientmay be from about 0 to 50% by weight, preferably from about 1 to 20% byweight, based on the total amount of the injection.

In a case where the sustained-release microsphere injection is dispersedin distilled water for injection or any appropriate dispersion medium atthe time of administration, the content of the microspheres may be fromabout 1 to 80% by weight, preferably from about 10 to 60% by weight,based on the total amount of the dispersion medium and the microspheres.

The microspheres of the present invention may be formulated into an oralpreparation according to any known method. For example, the microspheresof the present invention are mixed with an excipient (such as lactose,white sugar and starch), a disintegrator (such as starch and calciumcarbonate), a binder (such as starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, and hydroxypropyl cellulose), alubricant (such as talc, magnesium stearate and polyethylene glycol6000) or the like, compression-molded, and then, if necessary, coated byany known method for the purpose of masking the taste or giving entericor sustained-release property to obtain a oral preparation. Examples ofsuch a coating agent include hydroxypropylmethyl cellulose, ethylcellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetatephthalate, hydroxypropylmethyl cellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (manufactured by Rohm Company,Germany, methacrylic acid-acrylic acid copolymer), and a pigment such astitanium oxide and colcothar.

The microspheres produced according to the method of the presentinvention may be formulated into a nasal preparation in the form of asolid, semi-solid or liquid by any known method. For example, the solidnasal preparation may be made of the microspheres by themselves or maybe produced by adding and mixing an excipient (such as glucose,mannitol, starch, and microcrystalline cellulose), a thickener (such asnatural gum, a cellulose derivative and an acrylic acid polymer) or thelike to form a powdered composition. The liquid nasal preparation may beproduced as an oily or aqueous suspension in a similar manner to theabove injection. The semi-solid preparation is preferably produced as anaqueous or oily gel or an ointment form. These nasal preparations maycontain a pH regulator (such as carbonic acid, phosphoric acid, citricacid, hydrochloric acid, and sodium hydroxide), an antiseptic (such as apara-hydroxybenzoate ester, chlorobutanol and benzalkonium chloride) orthe like.

The microspheres of the present invention may be formulated into asuppository in the form of an oily or aqueous solid or semi-solid or aliquid according to any known method. An oily base used for the abovesuppository may be any oily base that does not allow the microsphere todissolve. Examples of such an oily base include glyceride of higherfatty acid [such as cacao butter and Witepsol-series products (DynamiteNobel)], medium fatty acid [such as Miglyol-series products (DynamiteNobel)], and a vegetable oil (such as sesame oil, soybean oil andcottonseed oil). Examples of an aqueous base include polyethyleneglycols and propylene glycol. Examples of aqueous gel base includenatural gums, cellulose derivatives, vinyl polymers, and acrylic acidpolymers.

The microspheres of the present invention are preferably used as aninjection.

The content of the microspheres of the present invention in thesustained-release composition of the present invention is preferably,but not limited to, at least about 70% by weight.

The microsphere of the present invention is less toxic and thus may beused as a safe pharmaceutical or the like for a mammal (such as human,bovine, swine, dog, cat, mouse, rat, and rabbit).

The dose of the microspheres of the present invention orsustained-release composition thereof may vary depending on the type andcontent of the physiologically active substance or a salt thereof as themain drug, the dosage form, the duration of release of thephysiologically active substance or a salt thereof, the target disease,the target animal, or the like, but may be set so as to provide aneffective amount of the physiologically active substance or a saltthereof. In a case where the sustained-release composition is a sixmonth preparation, for example, the dose of the physiologically activesubstance or a salt thereof as the main drug may be selected from therange of about 0.01 mg to 10 mg/kg, more preferably about 0.05 mg to 5mg/kg of body weight for an adult.

The dose of the microspheres may be selected from the range of about0.05 mg to 50 mg/kg, more preferably from about 0.1 mg to 30 mg/kg ofbody weight for an adult.

The frequency of administration may be once every several weeks, once amonth, once every several months (such as three, four or six months), orthe like and appropriately selected depending on the type and content ofthe physiologically active substance or a salt thereof as the main drug,the dosage form, the duration of release of the physiologically activesubstance or a salt thereof, the target disease, the target animal, orthe like.

The microsphere of the present invention or a sustained-releasecomposition thereof may be used as an agent for preventing or treatingvarious diseases depending on the type of the physiologically activesubstance or a salt thereof contained therein. In a case where thephysiologically active substance or a salt thereof is an LH—RHderivative, for example, the microsphere of the present invention or asustained-release composition thereof may be used as an agent forpreventing or treating a hormone-dependent disease, especiallyhormone-dependent cancer (such as prostatic cancer, uterus cancer,breast cancer, and pituitary tumor); a sex hormone-dependent diseasesuch as prostatic hypertrophy, endometriosis, hysteromyoma, precociouspuberty, dysmenorrheal, amenorrhea, premenstrual syndrome, andmultilocular ovarian syndrome; or such a disease as Alzheimer's diseaseand immunodeficiency; or may be used as an agent for contraception (orfor preventing or treating infertility, if a rebound effect is usedafter the drug holiday). The microsphere of the present invention or asustained-release composition thereof may also be used as an agent forpreventing or treating benign or malignant tumor that is not dependenton sex hormone but sensitive to LH—RH.

In order to produce the microspheres having improved dispersibilityaccording to the present invention, the osmotic pressure regulatingagent may be used in an outer aqueous phase when an emulsion containingthe physiologically active substance or a salt thereof and the polymeris subjected to in-water drying.

The present invention is more specifically described by means of thefollowing reference examples and examples, which are not intended tolimit the scope of the present invention.

EXAMPLES

In the following reference examples and examples, weight averagemolecular weight is determined in terms of polystyrene molecular weightby gel permeation chromatography (GPC) using monodisperse polystyrene asa reference material, and the content of each polymer is calculated fromeach weight average molecular weight. Each measurement is performed in ahigh performance GPC system (HLC-8120GPC manufactured by TosohCorporation) with SuperH4000×2 and SuperH2000 columns (each manufacturedby Tosoh Corporation) and a mobile phase of tetrahydrofuran at a flowrate of 0.6 ml/min. The detection is based on differential refractiveindex.

Reference Example 1 Synthesis of High Molecular Weight Lactic AcidPolymer

To 230 ml of dehydrated xylene were added 4.1 ml of a 1.0 mol/ldiethylzinc hexane solution, 1.35 g of tert-butyl lactate and 230 g ofDL-lactide and underwent polymerization reaction at 120 to 130° C. forabout two hours. After the reaction was completed, 120 ml ofdichloromethane was poured into the reaction liquid, and 230 ml oftrifluoroacetic acid was added to cause a deprotection reaction. Afterthe reaction was completed, 300 ml of dichloromethane was added to thereaction liquid, which was then poured into 2800 ml of isopropyl etherso that the desired product was precipitated. Re-precipitation withdichloromethane/isopropyl ether was repeated so that a lactic acidpolymer with a weight average molecular weight of about 40000 wasobtained.

Reference Example 2

The polymer obtained in Reference Example 1 was dissolved in 600 ml ofdichloromethane. After the resulting solution was washed with wateruntil it became neutral, 70 g of an aqueous 90% lactic acid solution wasadded and allowed to react at 40° C. When the weight average molecularweight of the polymer dissolved in the reaction liquid became about20,000, the reaction liquid was cooled to room temperature, and 600 mlof dichloromethane was poured to stop the reaction. The reaction liquidwas then washed with water until it became neutral. After the washingwith water, the reaction liquid was concentrated to dryness to give alactic acid polymer. In the resulting lactic acid polymer, the amount ofthe terminal carboxyl group was about 80 μmol per 1 g of the polymer andthe content of a polymer with a weight average molecular weight of 5000or less was 7.29% by weight.

Reference Example 3 (1)

The polymer obtained in Reference Example 1 was dissolved in 600 ml ofdichloromethane. After the resulting solution was washed with wateruntil it became neutral, 70 g of an aqueous 90% lactic acid solution wasadded and allowed to react at 40° C. When the weight average molecularweight of the polymer dissolved in the reaction liquid became about20,000, the reaction liquid was cooled to room temperature, and 600 mlof dichloromethane was poured to stop the reaction. After the reactionliquid was washed with water until it became neutral, the reactionliquid was added dropwise to 2800 ml of isopropyl ether so that thedesired lactic acid polymer was precipitated. The precipitate collectedby decantation was dissolved in 600 ml of dichloromethane. The resultingsolution was concentrated to dryness to give 160 g of a lactic acidpolymer. In the resulting lactic acid polymer, the amount of theterminal carboxyl group was about 70 μmol per 1 g of the polymer. Table1 shows the weight average molecular weights of the high molecularweight lactic acid polymers used, the weight average molecular weightsof the lactic acid polymers produced by the hydrolysis, and the weightaverage molecular weights and molecular weight distribution of theobtained target lactic acid polymer.

Reference Examples 3 (2) to (6)

Lactic acid polymers according to the present invention were obtained ina similar manner to Reference Example 3 (1). Table 1 shows the weightaverage molecular weights of the high molecular weight lactic acidpolymers used, the weight average molecular weights of the lactic acidpolymers produced by the hydrolysis, and the weight average molecularweights and molecular weight distribution of the obtained target lacticacid polymers. TABLE 1 Reference Example 3 (1) (2) (3) (4) (5) (6) Mw ofHigh Molecular 40500 43600 40400 43300 38600 55000 Weight Lactic AcidPolymer Mw of Hydrolysis 22200 22200 22700 22200 18600 27200 Product Mwof Obtained Lactic 22900 22200 21900 22300 19400 28200 Acid PolymerMolecular 1-1000 0.03 0.07 0.00 0.01 0.08 0.04 Weight 1-3000 0.95 1.120.87 0.09 1.45 0.62 Distribution 1-5000 3.86 4.17 3.89 3.92 4.89 2.50(%)

Table 1 indicates that in each lactic acid polymer produced by themethod according to the present invention, the content of a polymer witha weight average molecular weight of 5000 or less is at most about 5% byweight; the content of a polymer with a weight average molecular weightof 3000 or less is at most about 1.5% by weight; and the content of apolymer with a weight average molecular weight of 1000 or less is atmost about 0.1% by weight.

Comparative Example 1

In 354.3 g of dichloromethane was dissolved 205.5 g of a DL-lactic acidpolymer (with a weight average molecular weight of 21,400 and a carboxylamount of 76.1 μmol/g determined by labeling quantitative determination)which was obtained in a similar manner to Reference Example 3 (1). Theresulting solution was filtered under pressure with a 0.2 μm filter(DFA4201FRP, EMFLOW) and adjusted to 28.8° C. After 380.4 g of theresulting organic solvent solution was weighed out, it was mixed with anaqueous solution of 16.11 g of peptide A acetate in 16.22 g of distilledwater, which was previously heated to 55.4° C. The mixture was stirredfor 1 minute to be roughly emulsified and then emulsified at 10,150 rpmwith a mini mixer for 2 minutes to form a W/O emulsion. After cooled to18° C., the W/O emulsion was poured into 25 liters of an aqueous 0.1%(w/w) polyvinyl alcohol (EG-40 manufactured by The Nippon Synthetic)solution which was previously adjusted to 18.7° C. over 3 minutes and 10seconds and then stirred at 7,001 rpm with Homomic Line Flow(manufactured by Tokushu Kika Kogyo Co., Ltd.) to form a W/O/W emulsion.The temperature of the W/O/W emulsion was adjusted to about 18.5° C. for30 minutes and then stirred for 2 hours and 30 minutes withouttemperature adjustment so that dichloromethane and ethanol werevolatilized or diffused into the outer aqueous phase and that the oilphase was solidified. After passed through a 75 μm mesh sieve, themicrospheres were continuously precipitated and collected at 2,000 rpmwith a centrifuge (H-600S manufactured by Kokusan Corporation) Thecollected microspheres were dispersed in a small amount of distilledwater again and passed through a 90 μm mesh sieve. Thereto 18.85 g ofmannitol was added and dissolved. The mixture was lyophilized to obtainmicrosphere powder. The mass and the yield of the resulting microspherepowder were 117.6 g and 68.54% respectively. The content of peptide Awas 7.76%. An electron micrograph of the resulting microspheres is shownin FIG. 1.

Example 1

In 354.4 g of dichloromethane was dissolved 205.4 g of a DL-lactic acidpolymer (with a weight average molecular weight of 21,400 and a carboxylamount of 76.1 μmol/g determined by labeling quantitative determination)which was obtained in a similar manner to Reference Example 3 (1). Thetemperature of the resulting solution was adjusted to 30° C. After 380.5g of the resulting solution was weighed out, it was mixed with anaqueous solution of 16.1 g of leuprorelin acetate in 16.2 g of distilledwater, which was previously heated at 55° C. The mixture was emulsifiedwith a mini mixer (Tokushu Kika Kogyo Co., Ltd.) to form a W/O emulsion(at a rotation speed of about 10,000 rpm). After cooled to about 18° C.,the W/O emulsion was poured into 25 liters of an aqueous 0.1% (w/w)polyvinyl alcohol (EG-40 manufactured by The Nippon Synthetic)+1%mannitol solution which was previously adjusted to about 18° C., andthen secondarily emulsified with Homomic Line Flow (manufactured byTokushu Kika Kogyo Co., Ltd.) to form a W/O/W emulsion (at a turbinerotation speed of about 7,000 rpm and a circulating pump rotation speedof about 2000 rpm). The W/O/W emulsion was subjected to in-water dryingfor about 3 hours, passed through a standard 75 μm sieve, and thencentrifuged (H-600S manufactured by Kokusan Corporation) to precipitatecontinuously and collect microspheres (at a rotation speed of about2,000 rpm and a flow rate of about 600 ml/min). The collectedmicrospheres were dispersed in a small amount of distilled water againand passed through a standard 90 μm sieve. Thereto 18.9 g of mannitolwas added. The mixture was lyophilized with a lyophilizer (Triomastermanufactured by Kyowa Vacuum Engineering) to obtain powder (microspherepowder). An electron micrograph of the resulting microspheres is shownin FIG. 2.

Experimental Example 1

About 660 mg of the microsphere powder produced in Comparative Example 1or Example 1 was weighed in a coat 9P vial, which was then plugged witha rubber stopper and sealed with a screw cap. To the vial was added 1.5ml of a dispersion medium for leuprorelin acetate (a mixture of 5%mannitol, 1% carmellose sodium and 0.1% polysorbate 80), and the timerequired for uniform dispersion to be attained was measured.

Each microsphere powder was dispersed by shaking at a shaking width ofabout 7 cm and a shaking speed of about 30 times/10 seconds according toinstructions attached to a leuprorelin acetate vial preparation. Theresults are shown in Table 2. TABLE 2 Comparative Example 1 Example 1Dispersion Time about 2 to 4 minutes 8 to 23 seconds

Experimental Example 2

About 660 mg of the microsphere C powder produced in Comparative Example1 or Example 1 was charged into a 14φ type DPS (dual-chamber prefilledsyringe) which was filled with a dispersion medium for leuprorelinacetate (the amount of the dispersion liquid: 1.5 ml), and suspended.The time required for uniform dispersion to be attained was measured.

Each microsphere powder was dispersed by tapping the syringe on a palmat a shaking width of about 3 cm and at a shaking speed of about 50times/10 seconds according to instructions attached to a leuprorelinacetate DPS preparation. The results are shown in Table 3. TABLE 3Comparative Example 1 Example 1 Dispersion Time about 2 to 6 minutes 20to 46 secondsIndustrial Applicability

The microspheres of the present invention have improved dispersibilityand thus can be dispersed at a high concentration in a dispersion mediumsuch as distilled water for injection.

1. A method of producing a microsphere having improved dispersibility,which comprises adding an osmotic pressure regulating agent to an outeraqueous phase in producing the microspheres by an in-water dryingmethod.
 2. The method according to claim 1, wherein the dispersibilityis improved to such a degree that about 400 to about 700 mg of themicrospheres can be dispersed in 1.5 ml of a dispersion medium forinjection in less than two minutes.
 3. The method according to claim 1,wherein a W/O/W type emulsion is used in the in-water drying method. 4.The method according to claim 3, which further comprises adding a drugcarrier to an inner aqueous phase.
 5. The method according to claim 1,wherein an O/W type emulsion is used in the in-water drying method. 6.The method according to claim 1, wherein an S/O/W type emulsion is usedin the in-water drying method.
 7. A method of producing microspheres,which comprises dispersing a W/O type emulsion in an outer aqueous phasethat contains an osmotic pressure regulating agent, wherein the W/O typeemulsion consists of an inner aqueous phase containing a physiologicallyactive substance or a salt thereof and an oil phase of a solutioncontaining a lactic acid polymer with a weight average molecular weightof 15000 to 50000 or a salt thereof; and subjecting the dispersion to anin-water drying method.
 8. The method according to claim 7, wherein thecontent of a polymer with a weight average molecular weight of 5000 orless in the lactic acid polymer or the salt thereof is about 10% byweight or less.
 9. The method according to claim 7, wherein the contentof a polymer with a weight average molecular weight of 5000 or less inthe lactic acid polymer or the salt thereof is about 5% by weight orless.
 10. The method according to claim 7, wherein the content of apolymer with a weight average molecular weight of 3000 or less in thelactic acid polymer or the salt thereof is about 1.5% by weight or less.11. The method according to claim 7, wherein the content of a polymerwith a weight average molecular weight of 1000 or less in the lacticacid polymer or the salt thereof is about 0.1% by weight or less. 12.The method according to claim 7, wherein the weight average molecularweight of the lactic acid polymer or the salt thereof is 15000 to 40000.13. The method according to claim 7, wherein the weight averagemolecular weight of the lactic acid polymer or the salt thereof is 17000to
 26000. 14. The method according to claim 1, wherein the osmoticpressure regulating agent is alcohol, sugar, amino acid, a peptide, aprotein, a salt of water-soluble amino acid, or a derivative thereof ora mixture thereof.
 15. The method according to claim 1, wherein theosmotic pressure regulating agent is mannitol.
 16. The method accordingto claim 1, wherein a concentration of the osmotic pressure regulatingagent in the outer aqueous phase is a concentration at which the osmoticpressure of the outer aqueous phase is about 1/50 to about 5 times theosmotic pressure of isotonic sodium chloride solution.
 17. The methodaccording to claim 7, wherein the physiologically active substance is awater-soluble physiologically active substance.
 18. The method accordingto claim 7, wherein the physiologically active substance is aphysiologically active peptide.
 19. The method according to claim 7,wherein the physiologically active substance is an LH—RH derivative. 20.The method according to claim 7, wherein the LH—RH derivative is apeptide represented by the formula:5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z wherein Y represents DLeu,DAla, DTrp, DSer(tBu), D2Nal or DHis(ImBzl) and Z represents NH—C₂H₅ orGly-NH₂, or a salt thereof.
 21. A microsphere produced by the methodaccording to claim
 1. 22. A sustained-release composition comprising themicrosphere according to claim
 21. 23. The sustained-release compositionaccording to claim 22, which is for prevention or treatment of prostaticcancer, prostatic hypertrophy, endometriosis, hysteromyoma,metrofibroma, precocious puberty, dysmenorrhea or breast cancer, or forcontraception.
 24. The sustained-release composition according to claim22, which is for injection.
 25. The sustained-release compositionaccording to claim 22, which further comprises mannitol.
 26. Thesustained-release composition according to claim 22, which contains atleast about 70% by weight of the microsphere in the total composition.27. A method of preventing or treating prostatic cancer, prostatichypertrophy, endometriosis, hysteromyoma, metrofibroma, precociouspuberty, dysmenorrhea or breast cancer or of contraception, whichcomprises administering an effective amount of the sustained-releasecomposition according to claim 22 to a mammal.
 28. A method whichcomprises subjecting an emulsion to in-water drying in the presence ofan osmotic pressure regulating agent in the outer aqueous phase forproducing a microsphere having improved dispersibility, wherein theemulsion contains a physiologically active substance or a salt thereofand a polymer.
 29. Use of an osmotic pressure regulating agent in anouter aqueous phase in subjecting an emulsion containing aphysiologically active substance or a salt thereof and a polymer toin-water drying for production of a microsphere having improveddispersibility.